Comparative evaluation of nutrient media quality control using LYOSHTAMM lyophilised test strains: Results of interlaboratory trials

INTRODUCTION. Microbiological quality control of medicinal products relies on the use of reference test strains with stable cultural and morphological properties. Use of lyophilised test strains standardised by number of viable cells reduces the labour intensity of testing compared to the traditional method of preparing microbial suspensions.

AIM. This study aimed to evaluate reproducibility, homogeneity, and stability of results when assessing growth-promoting properties of nutrient media using traditional preparation method of microbial suspensions and the method based on LYOSHTAMM lyophilised test strains standardised by the number of viable cells.

MATERIALS AND METHODS. Two test strains were used: Salmonella enterica subsp. enterica serovar Abony IHE 103/39 and Staphylococcus aureus ATCC 6538. Compared was the method using standardised lyophilised LYOSHTAMM samples (one batch per strain, standardised by the number of viable cells) and the reference method. The latter uses microbial suspensions prepared in compliance with the Russian State Pharmacopoeia (pharmacopoeial monograph “Microbiological purity”). The study was conducted across seven independent laboratories over three days. A total of 378 individual results were obtained for each LYOSHTAMM batch and 126 results — for the reference method.

RESULTS. When evaluating growth-promoting properties of the media, both test strains based on LYOSHTAMM samples met all acceptability criteria: the number of colony-forming units per plate ranged from 10 to 100; typical cultural and morphological properties and growth time was confirmed. Intralaboratory precision (RSD) ranged from 7% to 21% for LYOSHTAMM method and from 2% to 36% for reference method. The interlaboratory precision for LYOSHTAMM samples was 15–17%, while for reference method, it was 38–45%. Time to visible colonies (≈18 hours) and their cultural and morphological properties showed no significant differences between the methods. Analysis of inter-vial homogeneity showed consistent mean values and relative standard deviations at intralaboratory production control (n=5) and  interlaboratory trials (n=63), confirming the stability of LYOSHTAMM batch (relative standard deviation ≤17%).

CONCLUSIONS. The control method for nutrient media using standardised lyophilised LYOSHTAMM test strains yields results comparable to the traditional method in  terms  of growth and morphology. A key advantage of this method is a significantly improved interlaboratory reproducibility, with a more than two-fold reduction in variability. LYOSHTAMM test strains can be recommended as reference standards for routine microbiological testing and analytical validation.

1. Gupta D, Bhatt S, Shulka P, Kumar A. Microbial contamination in pharmaceutical manufacturing. J Drug Discov Health Sci. 2024;1(01):21–7. https://doi.org/10.21590/yafz5353

2. Tyski S, Burza M, Laudy AE. Microbiological contamination of medicinal products — Is it a significant problem? Pharmaceuticals (Basel). 2025;18(7):946. https://doi.org/10.3390/ph18070946

3. de Sousa Lima CM, Fujishima MAT, de Paula Lima B, et al. Microbial contamination in herbal medicines: a serious health hazard to elderly consumers. BMC Complement Med Ther. 2020;20(1):17. https://doi.org/10.1186/s12906-019-2723-1

4. Han-Min O. Selection of bacterial strains in a testing microbiology laboratory for quality assurance purposes: ISO/ IEC 17025: 2017 standard point of view. Accred Qual Assur. 2025;30:95–101. https://doi.org/10.1007/s00769-024-01615-9

5. Moira DJ. A simple and rapid test for quality control of liquid media, using the bioscreen microbiological growth analyser. J Microbiol Methods. 1998;32(1):37–43. https://doi.org/10.1016/S0167-7012(98)00007-4

6. Sukhanova SM. Standardization of requirements for culture media used to assess the quality of drugs (Review). Drug Development & Registration. 2023;12(1):123–30 (In Russ.). https://doi.org/10.33380/2305-2066-2023-12-1-123-130

7. Kuleshova SI, Protsak SA, Lisunova SA, Romanyuk GYu. Stability of ready-to-use and laboratory-prepared culture media. Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products. 2021;11(2):130–4 (In Russ.). https://doi.org/10.30895/1991-2919-2021-11-2-130-134

8. Basu S, Pal A, Desai PK. Quality control of culture media in a microbiology laboratory. Indian J Med Microbiol. 2005;23(3):159–63. https://doi.org/10.1016/S0255-0857(21)02585-8

9. Moser AI, Campos-Madueno EI, Perreten V, Endimiani A. Genome stability during serial subculturing in hyperepidemic multidrug-resistant Klebsiella pneumoniae and Escherichia coli. J Glob Antimicrob Resist. 2022;31:152–61. https://doi.org/10.1016/j.jgar.2022.08.014

10. Wassenaar TM, Zimmermann K. How industrial bacterial cultures can be kept stable over time. Lett Appl Microbiol. 2020;71(3):220–8. https://doi.org/10.1111/lam.13309

11. Sabol A, Joung YJ, Van Tubbergen C, et al. Assessment of genetic stability during serial in vitro passage and in vivo carriage. Foodborne Pathog Dis. 2021;18(12):894–901. https://doi.org/10.1089/fpd.2021.0029

12. Singh KA, Rai R, Nair SS. Review on development of assigned value microbiological reference materials used in food testing. Food Microbiol. 2022;102:103904. https://doi.org/10.1016/j.fm.2021.103904

13. Verdonk GP, Willemse MJ, Hoefs SG, et al. The most probable limit of detection (MPL) for rapid microbiological methods. J Microbiol Methods. 2010;82(3):193–7. https://doi.org/10.1016/j.mimet.2010.04.012

14. Kurniawan YN, Yuji S, Koji S. Evaluation of a microbiological reference standard (EZ-AccuShotTM) as test material for proficiency testing (PT) scheme in the breweries. J Am Soc Brew Chem. 201876(4):219–22. https://doi.org/10.1080/03610470.2018.1502010

15. Hong SK, Choi SJ, Shin S, et al. Establishing quality control ranges for antimicrobial susceptibility testing of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus: a cornerstone to develop reference strains for Korean clinical microbiology laboratories. Ann Lab Med. 2015;35(6):635–8. https://doi.org/10.3343/alm.2015.35.6.635

16. Liu K, Ma W, Jin G. Research progress on improving the survival rate and extending the storage period of lactic acid bacteria through vacuum freeze-drying. Food Sci. 2024, 45(2):325–33. https://doi.org/10.7506/spkx1002-6630-20230801-002

17. Dias FRS, Lourenço FR. Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests. J Microbiol Methods. 2021;189:106312. https://doi.org/10.1016/j.mimet.2021.106312

18. Bernal-Mercado AT, Gutierrez-Pacheco MM, EncinasBasurto D, et al. Synergistic mode of action of catechin, vanillic and protocatechuic acids to inhibit the adhesion of uropathogenic Escherichia coli on silicone surfaces. J Appl Microbiol. 2020;128(2):387–400. https://doi.org/10.1080/03610470.2018.1502010

19. Boyd P, Eaton S, Magee J. The stability of micro-organisms preserved in LENTICULE discs, demonstrated by repeat sample distributions of the EQUAL Scheme for Indicator Organisms (water microbiology). J Appl Microbiol. 2006;100(2):272–8. https://doi.org/10.1111/j.1365-2672.2005.02774.x

20. Kupletskaya MB, Netrusov AI. Viability of lyophilized microorganisms after 50-year storage. Microbiology. 2011;80(6):850–3. https://doi.org/10.1134/S0026261711060129

21. Miyamoto-Shinohara Y, Sukenobe J, Imaizumi T, Nakahara T. Survival curves for microbial species stored by freezedrying. Cryobiology. 2006;52(1):27–32. https://doi.org/10.1016/j.cryobiol.2005.09.002

22. Morgan CA, Herman N, White PA, Vesey G. Preservation of micro-organisms by drying; a review. J Microbiol Methods. 2006;66(2):183–93. https://doi.org/10.1016/j.mimet.2006.02.017

23. Voropaev AA, Krysanova YuI, Fadeikina OV, et al. Lyophilization of microorganisms standardized by the number of viable cells in low concentration: Development of a drying mode. Biological Products. Prevention, Diagnosis, Treatment. 2025;25(2):193–202 (In Russ.). https://doi.org/10.30895/2221-996X-2025-25-2-193-202